Sequences 5' to the conserved 28 bp Leg box element regulate the expression of pea seed storage protein gene legA

The function of the 28 bp conserved Leg box element found in the 11S legumin-like genes of Vicia, Pisum, Glycine and Helianthus in controlling gene expression has been examined. Transgenic tobacco plants containing 124 bp of 5' legA flanking sequence including the Leg box and all sequences downstream to the start of transcription failed to activate the gene, as judged by ELISA and northern assays. Expression was first detected in a promoter truncation construct which contained 549 bp of 5' flanking sequence. These two promoter truncation constructs were additionally examined for their ability to interact with pea seed nuclear proteins. The use of DNA-protein binding and mobility shift assays showed that the -124 bp fragment which included the Leg box did not bind nuclear proteins. In contrast, nuclear proteins were shown to strongly bind to the -549 bp flanking sequence fragment. The correlation of transgenic plant analysis with DNA binding assays indicates that the highly conserved 28 bp Leg box element is not the sole promoter determinant in legumin gene expression.